Limited resources of fossil fuels and increasing amounts of CO2 released from them and causing the greenhouse phenomenon have raised a need for using biomass as a renewable and clean source of energy. One promising, alternative technology is the production of biofuels, such as ethanol, butanol or propanol from cellulosic materials. In the transportation sector biofuels are for the time being the only option, which could reduce the CO2 emissions by an order of magnitude. The ethanol can be used in existing vehicles and distribution systems and thus it does not require expensive infrastructure investments. Sugars derived from cellulosic and lignocellulosic renewable raw materials can also be used as raw materials for a variety of chemical products that can replace oil-based chemicals.
Most of the carbohydrates in plants are in the form of lignocellulose, which essentially consists of cellulose, hemicellulose and lignin. In a conventional lignocellulose-to-ethanol process the lignocellulosic material is first pretreated either chemically or physically, using acid hydrolysis, steam explosion, ammonia fiber expansion, alkaline wet oxidation or ozone pretreatment, to make the cellulose fraction more accessible to enzymatic hydrolysis. The cellulose fraction is then hydrolyzed to obtain sugars that can be fermented by yeast into ethanol and distilled to obtain pure ethanol. Lignin is obtained as a main co-product that may be used as a solid fuel. In this separate hydrolysis and fermentation (SHF) process the temperature of enzymatic hydrolysis is typically higher than that of fermentation. The use of thermostable enzymes in hydrolysis offer potential benefits, such as higher reaction rates at elevated temperatures, reduction of enzyme load due to higher specific activity and life-time of enzymes, increased flexibility with respect to process configuration and better hygiene.
There is continuous research for making the bioethanol production process more economical. One of the options is the simultaneous saccharification and fermentation (SSF) process. The principal benefits are the reduced end-product inhibition of the enzymatic hydrolysis and the reduced investment costs. The challenges are in finding favorable conditions, e.g. temperature and pH, for both the enzymatic hydrolysis and fermentation. In the consolidated bioprocess (CBP), the amount of externally added enzymes can be significantly reduced by exploiting a fermentative organism or ethanolgen, which is capable of producing a set of lignocellulolytic enzymes.
In recent years, metabolic engineering for microorganisms used in ethanol production has shown significant progress. Besides Saccharomyces cerevisiae, microorganisms such as the bacterial species Zymomonas and Escherichia coli and yeasts such as Pichia stipitis and Kluyveromyces fragilis have been targeted for ethanol production from cellulose. In the SSF process, the inhibitor and temperature tolerance as well as the ability to utilize multiple sugars are important properties of the fermenting microorganism. Engineered yeasts have been developed that are able to ferment pentose sugars xylose and arabinose in addition to glucose. Thermophilic microbes, like Thermoanaerobacterium saccharolyticum or Clostridium thermocellum have been engineered to ferment sugars, including xylose, to ethanol at elevated temperatures of 50° C.-60° C. (thermophilic SSF or TSSF). Such fermentative organisms have also potential as CBPs (Shaw et al. 2008).
Enzymatic hydrolysis is considered the most promising technology for converting cellulosic biomass into fermentable sugars. However, enzymatic hydrolysis is used only to a limited amount at industrial scale, and especially when using strongly lignified material such as wood or agricultural waste the technology is not satisfactory. Efforts have been made to improve the efficiency of the enzymatic hydrolysis of the cellulosic material (Badger 2002; Kurabi et al., 2005).
WO2001060752 (Forskningscenter Risø, DK) describes a continuous process for converting solid lignocellulosic biomass into combustible fuel products. After pretreatment by wet oxidation or steam explosion the biomass is partially separated into cellulose, hemicellulose and lignin, and is then subjected to partial hydrolysis using one or more carbohydrase enzymes (EC 3.2).
WO2002024882 (Iogen Bio-Products Corp., CA) pertains a method of converting cellulose to glucose by treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase and a modified cellobiohydrolase I (CBHI) obtained by inactivating its cellulose binding domain (CBD). US2004/0005674 (Athenix Corp., US) describes novel enzyme mixtures that can be used directly on lignocellulose substrate. The synergistic enzyme mixture contains a cellulase and an auxiliary enzyme such as cellulase, xylanase, ligninase, amylase, protease, lipidase or glucuronidase, or any combination thereof. Cellulase is considered to include endoglucanase (EG), beta-glucosidase (BG) and cellobiohydrolase (CBH) enzymes.
US20050164355 (Novozymes Biotech Inc., US) describes a method for degrading lignocellulosic material with one or more cellulolytic enzymes selected from EG, BG and CBH and in the presence of at least one surfactant. Additional enzymes such as hemicellulases, esterase, peroxidase, protease, laccase or mixture thereof may also be used.
The best-investigated and most widely applied cellulolytic enzymes of fungal origin have been derived from Trichoderma reesei (the anamorph of Hypocrea jecorina). Cellulases from less known fungi have also been disclosed.
Hong et al. (2003a and 2003b) characterize EG and CBHI of Thermoascus aurantiacus and their production in yeast. Tuohy et al. (2002) describe three forms of cellobiohydrolases, including CBHI and CBHII from Talaromyces emersonii. 
Use of cellobiohydrolase I (CBHI), a member of family 7 of glycosyl hydrolases in enzymatic conversion of cellulosic material is known, for example from WO03/000941 (Novozymes A/S, DK), which relates to CBHI enzymes obtained from various fungi. WO2005074656 (Novozymes Inc., US) discloses polypeptides having cellulolytic activity derived e.g. from Thermoascus aurantiacus. 
WO2007071818 (Roal Oy, FI) describes production of sugar hydrolysates from cellulosic material by enzymatic conversion and enzyme preparations comprising said enzymes. Enzymes useful in the method include thermostable cellobiohydrolase, endoglucanase, beta-glucosidase and optionally xylanase deriving from Thermoascus aurantiacus, Acremonium thermophilum or Chaetomium thermophilum. 
Cellobiohydrolases II have been disclosed in several applications. WO2004056981 (Novozymes A/S, DK) discloses polypeptides having cellobiohydrolase II activity and polynucleotides encoding the polypeptides as well as methods for producing and using the polypeptides in applications, such as in production of ethanol. Full length DNA sequences are disclosed from Aspergillus tubigensis, Chaetomium thermophilum, Myceliophtora thermophila, species of Thielavia, Acremonium thermophilum, Trichophaea saccata, Stibella anualata and Malbrancheae cinnamonea. EP1578964 B1 (Novozymes A/S, DK) discloses the full length amino acid sequence of C. thermophilum CBHII and a polypeptide encoded by a nucleotide sequence hybridizing under stringent conditions with a fragment of the nucleotide sequence encoding said enzyme.
CN1757709 (Shandong Agricultural Univ., CN) discloses the nucleotide sequence of a thermophilic CBHII enzyme of Chaetomium thermophilum CT2 and its expression in Pichia pastoris yeast. The enzyme is capable of converting the rejected fiber material.
WO2006074005 (Genencor Int., Inc., US) discloses a variant of Hypocrea jecorina (Trichoderma reesei) CBHII/Cel6A enzyme. The variant enzyme is useful, for example in bioethanol production.
WO2007094852 (Diversa Corp., US; Verenium Corp., US) discloses cellulolytic enzymes, nucleic acids encoding them and methods for their production and use. The enzyme may be an endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanase, a beta-xylosidase, an arabinofuranosidase or an oligomerase. The enzyme and enzyme mixtures are useful, for example in making fuel or bioethanol.
WO2008095033 (Syngenta, CH, Verenium Corp., US) discloses enzymes having lignocellulolytic activity, including cellobiohydrolases useful, for example in making fuels and processing biomass materials. WO2009045627 (Verenium Corp., US) discloses methods for breaking down hemicellulose by using enzymes having xylanase, mannanase and/or glucanase activity and increased activity and stability at increased pH and temperature.
WO2009089630 (Iogen Energy Corp., CA) discloses a variant of a family 6 cellulase with reduced inhibition by glucose, comprising one or more amino acid substitutions.
WO2009059234 (Novozymes Inc., US) discloses methods of producing cellulosic material reduced in a redox active metal ion useful in degrading or converting a cellulosic material and producing a fermentation product. The application discloses, e.g. a CBHII polypeptide of Chaetomium thermophilum. 
WO2009085868 (Novozymes A/S, DK) discloses polypeptides, polynucleotides encoding the enzyme and a method for producing a fermentation product, comprising saccharification of a cellulosic material with a cellulolytic enzyme composition comprising said polypeptide. Such cellobiohydrolases may derive from Trichoderma reesei, Humicola insolens, Myceliophtora thermophila, Thielavia terrestris and Chaetomium thermophilum. 
WO2006074435 and US2006218671 (Novozymes Inc., US) disclose nucleotide and amino acid sequences of Thielavia terrestris Cel6A cellobiohydrolase. U.S. Pat. No. 7,220,565 (Novozymes Inc., US) discloses polypeptides having cellulolytic enhancing activity and identity to the mature amino acid sequence of Myceliophtora thermophila CBHII.
US20070238155 and US20090280105 (Dyadic Int., Inc., US) disclose enzyme compositions comprising novel enzymes from Chrysosporium lucknowense, comprising e.g. the CBHIIa and CBHIIb enzymes assigned to family 6 of glycosyl hydrolases. The enzyme compositions are effective in hydrolysis of the lignocellulosic material.
The genome sequence of Neurospora crassa OR74A is disclosed in Galagan et al. (2003), including a sequence of exoglucanase 2 precursor. Collins et al. (2003) disclose the coding sequence of Talaromyces emersonii CBHII/Cel6A.
The market in biofuels such as renewable transportation fuels is expected to increase considerably in near future. As a result, there is a rapidly growing interest in the use of alternative feedstock for biofuel production. Fermentation of cellulosic biomass present in plants and woods or municipal waste to ethanol and other alcohols is an attractive route to fuels that supplement fossil fuels. One barrier of production of biofuels from cellulosic and lignocellulosic biomass is the robustness of the cell walls and the presence of sugar monomers in the form of inaccessible polymers that require a great amount of processing to make the sugar monomers available to the microorganisms that are typically used to produce alcohol by fermentation. Thus, there is a continuous need for new methods as well as new enzymes and enzyme mixtures, which enhance the efficiency of the degradation of the cellulosic and lignocellulosic substrates. Particularly, enzymes and enzyme mixtures are needed which are able to attack different glycosidic linkages of the crystalline cellulosic material and thus provide almost complete hydrolysis of the varying materials to be treated. There is also a need for enzymes, which are stable at elevated process temperatures, thus enabling the use of high biomass consistency and leading to high sugar and ethanol concentrations. This approach may lead to significant savings in energy and investments costs. The high temperature also decreases the risk of contamination during hydrolysis. The present invention aims to meet at least part of these needs.